DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE
839
Development 138, 839-848 (2011) doi:10.1242/dev.061804
© 2011. Published by The Company of Biologists Ltd
Brassinosteroid perception in the epidermis controls root
meristem size
Yael Hacham1, Neta Holland1, Cristina Butterfield2, Susana Ubeda-Tomas3, Malcolm J. Bennett3,
Joanne Chory2,4 and Sigal Savaldi-Goldstein1,2,*
SUMMARY
Multiple small molecule hormones contribute to growth promotion or restriction in plants. Brassinosteroids (BRs), acting
specifically in the epidermis, can both drive and restrict shoot growth. However, our knowledge of how BRs affect meristem size
is scant. Here, we study the root meristem and show that BRs are required to maintain normal cell cycle activity and cell
expansion. These two processes ensure the coherent gradient of cell progression, from the apical to the basal meristem. In
addition, BR activity in the meristem is not accompanied by changes in the expression level of the auxin efflux carriers PIN1, PIN3
and PIN7, which are known to control the extent of mitotic activity and differentiation. We further demonstrate that BR
signaling in the root epidermis and not in the inner endodermis, quiescent center (QC) cells or stele cell files is sufficient to
control root meristem size. Interestingly, expression of the QC and the stele-enriched MADS-BOX gene AGL42 can be modulated
by BRI1 activity solely in the epidermis. The signal from the epidermis is probably transmitted by a different component than
BES1 and BZR1 transcription factors, as their direct targets, such as DWF4 and BRox2, are regulated in the same cells that express
BRI1. Taken together, our study provides novel insights into the role of BRs in controlling meristem size.
INTRODUCTION
The control of final cell and organ size is a fundamental question
in the biology of all multicellular organisms. Root length is
determined by the number of proliferating cells and their mature
final size (Beemster and Baskin, 1998). The root is characterized
by consecutive developmental zones along its proximal-distal axis.
These zones form a gradient of renewed cells that proliferate,
elongate and differentiate (Fig. 1A). In the root meristem zone,
cells undergo repeated rounds of cell division. Subsequently, the
cells exit the meristematic zone to become part of the
elongation/differentiation zone (EDZ) where cells cease dividing,
undergo rapid cell expansion and differentiate. The root
meristematic zone can be further divided into the apical and basal
meristem zones (Beemster et al., 2003; Ishikawa and Evans, 1995;
Verbelen et al., 2006; Zhang et al., 2010). The apical meristem is
characterized by a high rate of cell proliferation, where cells do not
exhibit a significant gain in size. In the basal meristem, also
referred to as the transition zone between the apical meristem and
the elongation zone, cell proliferation rate slows or stops and cells
become larger. The quiescent center (QC, organizing cells),
together with their surrounding stem cells, define the stem cell
niche.
Brassinosteroids (BRs) are essential for normal plant growth and
development, and mutants that are unable to synthesize or perceive
BRs are dwarfs. BRs are perceived upon direct binding to the
extracellular domain of the cell surface receptor kinase BRI1 (He
1
Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel. 2Plant
Biology Laboratory, The Salk Institute, La Jolla, CA 92037, USA. 3Centre for Plant
Integrative Biology, University of Nottingham, Nottingham LE12 5RD, UK. 4Howard
Hughes Medical Institute, The Salk Institute, La Jolla, CA 92037, USA.
*Author for correspondence ([email protected])
Accepted 14 December 2010
et al., 2000; Li and Chory, 1997). The signal is then transmitted
from the plasma membrane to the nucleus, where
dephosphorylation of the transcription factors, BES1 and BZR1,
allows them to homo- or hetero-dimerize and bind DNA to regulate
the expression of hundreds of genes (He et al., 2005; Kim et al.,
2009; Yin et al., 2005). BES1 and BZR1 induce or repress the
expression of their direct-target genes upon binding to two
identified cis-elements, E-BOX and BRRE. The latter is found in
many genes, including the BR-biosynthesis genes, which undergo
rapid inhibition by BZR1 in response to BRI1 activation (He et al.,
2005).
Several studies have attributed the growth defects of BR mutants
primarily to impaired cell expansion (Clouse and Sasse, 1998;
Perez-Perez et al., 2002; Savaldi-Goldstein et al., 2007; Szekeres
et al., 1996), with a smaller effect on cell division (Mouchel et al.,
2004; Mouchel et al., 2006; Nakamura et al., 2006; Nakaya et al.,
2002; Reinhardt et al., 2007). However, our knowledge of how
BRs regulate root growth and meristem size is scant and systematic
analysis is lacking.
Multiple phytohormones contribute to the regulation of root
growth. Auxin gradients, which are set up by the action of PIN
auxin efflux carriers, control the extent of mitotic activity and
differentiation (Galinha et al., 2007; Grieneisen et al., 2007).
Cytokinins promote cell differentiation by inducing the expression
of SHY2, a negative regulator of the expression of several PIN
genes (Dello Ioio et al., 2008). Gibberellins (GA) promote cell
expansion and the rate of cell proliferation through downregulation
of cell cycle inhibitors (Achard et al., 2009; Ubeda-Tomas et al.,
2009; Ubeda-Tomas et al., 2008). Finally, ethylene inhibits cellelongation through interaction with auxin (Ruzicka et al., 2009;
Stepanova et al., 2005; Swarup et al., 2007).
Recent works have shown that the activity of plant hormones
from a subset of cells can control growth and development of the
entire organ (Dello Ioio et al., 2007; Savaldi-Goldstein et al., 2007;
DEVELOPMENT
KEY WORDS: Arabidopsis, Brassinosteroids, Growth, Cell proliferation, Cell-cell communication
RESEARCH ARTICLE
Swarup et al., 2001; Swarup et al., 2005; Swarup et al., 2007;
Ubeda-Tomas et al., 2009; Ubeda-Tomas et al., 2008). For
example, BR signaling in the epidermis was found to both drive
and restrict shoot growth (Savaldi-Goldstein et al., 2007). However,
how BRs control meristem size remains unsolved. To address this
issue, Arabidopsis roots were chosen as they represent a simplified
developmental system, owing to a large number of available cell
marker lines and the well-described radial organization of cell files
that are accessible to imaging (Fig. 1A) (Petricka and Benfey,
2008).
Here, we show that the small meristem size of bri1 roots is
attributed to both an impaired cell cycle activity and cell expansion.
These defects result in a failure of cells to progress normally from
the apical to the basal meristem. We further demonstrate that the
size of the root meristem is controlled by BRI1 activity in the
epidermis. Thus, when present in the epidermis, BRI1 initiates a
signal which regulates gene expression of the meristematic inner
cell files, i.e. AGL42. This signal is transmitted by a different
component from BES1 and BZR1, which regulate their direct
target genes locally. Taken together, we demonstrate the unique role
of BRs in the control of root meristem size and suggest that cellcell communication from the epidermis to the inner-meristematic
cells is involved.
MATERIALS AND METHODS
Plant material, growth conditions and chemical treatments
All Arabidopsis thaliana lines and bri1-116 are in the Columbia (Col-0)
background. Transgenic lines harboring the following transgenes have
been described previously: pPLTs-erCFP (Galinha et al., 2007), pWOX5erGFP and pSCR-H2B-YFP, pAGL42-GFP (Nawy et al., 2005); pPIN1PIN1-GFP (Benkova et al., 2003) and CycB1;1-GFP (Ubeda-Tomas et al.,
2009). Seeds were sterilized using a bleach solution with 1% hydrochloric
acid and plated on 0.5⫻ Murashige-Skoog medium (0.5 MS) (Duchefa
Biochemie) supplemented with 0.8% (wt/vol) plant agar (Duchefa
Biochemie). Plates were stratified in the dark at 4°C for 3 days and then
transferred to 22°C in cycles of 16 hours light (~50 mol m–2 s–1)/8 hours
dark for 5 to 7 days. For chemical treatments, the BR biosynthesis
inhibitor, BRZ, and BL were dissolved in 100% dimethyl sulfoxide
(DMSO). BRZ was added at a final concentration of 2 M or 3 M
as indicated. BL was added to liquid 0.5 MS at final concentration of
100 nM.
Vector constructs and transgenic lines
Plants were transformed by the standard floral dip method, using
Agrobacterium containing the pMLBART binary vector (Gleave, 1992).
Promoter fragments upstream of GL2-, SCR-, SHR-, DWF4- and RCH1coding sequences were amplified from genomic DNA or from plasmid
DNA (RCH1) (Dello Ioio et al., 2007) and cloned to the polylinker of
pBJ36. Primer sequences used for amplification and the corresponding
restriction sites for pBJ36 insertion are listed in Table S1 in the
supplementary material). BRI1-GFP was then cloned as a KpnI/KpnI
fragment into pBJ36-containing promoters. For pDWF4-GUS, the cloning
procedure is as described by Savaldi-Goldstein et al. (Savaldi-Goldstein et
al., 2007), except that a 3.7 kb promoter fragment was used. pGL2BES1D-GFP gene was cloned as described previously (Savaldi-Goldstein
et al., 2007), except that GL2 promoter fragment was used. Transgenic
lines were selected for BASTA resistance. The homozygous bri1
background was verified using CAPS marker digest with PmeI.
Root growth analysis
For root elongation measurements, seedlings were grown vertically for 7
days. Starting from day 2 after germination, seedlings were scanned every
24 hours and root length was measured using Image J software. Meristem
length and meristem cell number were determined according to the cortical
cells, from confocal microscopy images. Kinematic analysis of the relative
elongation rate (RLER) was performed as described previously (Beemster
Development 138 (5)
and Baskin, 2000). Two-tailed t-tests comparing transgenic lines with Col0 were performed using Microsoft Excel software (P≤0.05, see Tables S2S5 in the supplementary material).
Confocal microscopy
Fluorescence signals were detected using LSM 510 META confocal laserscanning microscope (Zeiss) with a 25⫻ water immersion objective lens
(N.A. 0.8). Roots were imaged in water supplemented with propidium
iodide (PI, 10 g/ml). PI, GFP, CFP and YFP were excited with the 561,
488, 458 and 514 nm laser, respectively. The fluorescence emission was
collected at 575 nm for PI, between 500 and 530 nm band-pass for GFP,
between 469 and 522 nm band-pass for CFP, and between 522 and 576 nm
band-pass for YFP.
RNA extraction and expression analysis
For RNA extraction, seedlings were grown horizontally in 0.5 MS plates.
After germination, paper stripes soaked with liquid 0.5 MS containing
DMSO (mock) or 100 nM BL were placed on the roots for 3 and 16 hours.
For the BRZ treatment, seedlings were grown in 0.5 MS plates
supplemented with 2 M BRZ. Root tip were cut (~0.2-0.5 cm from the
tip) and subjected to total RNA extraction using the Spectrum plant total
RNA kit (Sigma). Quantitative real-time PCR assays were performed in the
7300 real-time PCR system (Applied Biosystems, USA). Table S1 in the
supplementary material lists all the primer sequences. To normalize the
variance among samples, the At5g15400 transcript level was used as
endogenous control. Relative expression values were calculated using the
comparative Ct method with ABI Prism 7300 system SDS v.1.4
software (Applied Biosystems, USA). Detail on the Ct method is
available upon request. The values presented are the mean of two or three
biological replicates, each with three technical replicates. Bars indicate
s.e.m.
Histochemical assay and light microscope
GUS assays were performed as described (Savaldi-Goldstein et al., 2008),
except that the reaction was stopped by replacing the staining solution with
0.24 M HCl in 20% methanol, followed by the root clearance protocol
described previously (Malamy and Benfey, 1997). For sectioning, stained
seedlings were fixed in 1.25% glutaraldehyde, dehydrated, stained in 0.1%
Eosin and embedded in JB4 (EMS, USA) and sectioned (2-3 m). Intact
roots and root sections were analyzed by AxioImager (Zeiss).
Immunofluorescence analysis
Immunofluorescence of KNOLLE and PIN7 proteins was performed as
described previously (Muller et al., 1998). Five-day-old Arabidopsis
seedlings were fixed, incubated with anti- KNOLLE (1:2000) or anti-PIN7
(1:50) antibodies and then with secondary antibodies [Alexa Flour 568
donkey anti-rabbit (1:500) and Cy3 donkey anti-mouse (1:200),
respectively]. For nuclear staining, seedlings were incubated with 1 g/ml
DAPI solution for 10 minutes and washed three times with PBS buffer.
RESULTS
BRs control root length by promoting cell
expansion and maintaining normal cell number in
the root meristem
Root length is a result of integrated cell proliferation and cell
expansion rates. To determine to what extent these processes are
controlled by BR activity, we first characterized the rate of root
elongation in bri1 and wild-type seedlings. As shown in Fig. 1B,C,
wild-type seedlings continuously accelerated their root growth rate
between days 2 to 6 after germination, and this acceleration slowed
down after day 6 (Fig. 1C). By contrast, the roots of bri1 elongated
at a lower rate with a delayed small acceleration between days 5
and 6. As a result, at day 7, the root of bri1 is approximately onethird the length of wild type. Similar behavior was obtained in
measuring the meristem size (see Fig. S1A in the supplementary
material). We next calculated the relative rate of cell expansion
along the root [relative elongation rate (RLER)] for wild-type and
DEVELOPMENT
840
Epidermal BRI1 controls root growth
RESEARCH ARTICLE
Fig. 1. BRs control root length by promoting cell expansion rate.
(A)Confocal laser scanning microscope showing tissue organization and
developmental zones of Arabidopsis wild-type root. The root meristem is
subdivided into two developmental zones that are determined according
to the cortex cells. The apical meristem is characterized by high rate of
cell division and extends from the quiescent center (QC) to the first
notable larger cortical cell; basal meristem starts from the end of apical
meristem and ends at the start of elongation/differentiation zone (EDZ),
where cells exceed more than twice their size, stop dividing and, hence,
commence to differentiate. Cell borders are marked by PI (red). st, stele;
en, endodermis; c, cortex; ep, epidermis; lrc, lateral root cap.
(B,C)Reduced root length (B) and growth rate (C) of bri1 compared with
wild type. DAG, days after germination. (D)Kinematic analysis
quantifying the relative elongation rate (RLER) along bri1 mutant (black
circles) and wild-type (white circles) roots. (E)Direct measurement of
mature cortical cells length. Cell length is lower in bri1 than in wild type.
Data are mean±s.e.m.
bri1 (Beemster and Baskin, 2000; Ubeda-Tomas et al., 2008). The
maximum elongation rate of cells in bri1 was lower than in wild
type, and bri1 cells ceased elongating at 500 M from the root tip
(Fig. 1D). Indeed, mature cortex cells in bri1 mutants were
approximately half the length of wild type (Fig. 1E).
To test whether cell proliferation is also affected in bri1, we
measured both the size of the meristem and the number of
meristematic cells present in bri1 and wild type. The meristem length
was reduced in bri1 (Fig. 2A, left-most panel). In addition, when
841
Fig. 2. BRs promote cell expansion and cell number in the root
meristem. (A)Direct measurement of the length of the different root
zones. (B)Quantification of cortical cell number present in each zone and
the whole meristem. (C)Calculation of the average cortex cell length for
each meristematic zone. Seven-day-old seedlings were analyzed. Results
are presented as mean±s.e.m. Note the dramatic drop in the number of
cells and in their size in the basal meristem of bri1, relative to the apical
meristem, when compared with wild type.
compared with wild type, bri1 had 20% fewer meristematic cells
(Fig. 2B, left-most panel). The results show that BRs impact on
meristematic cell number in addition to promoting cell expansion.
BRs are required for normal cell cycle activity
We next asked whether the reduced cell number observed in the
root meristem of bri1 is a result of impaired cell cycle progression.
Therefore, we used the G2/M phase cell cycle marker CycB1;1GFP to monitor the occurrence of cell divisions (Colon-Carmona
et al., 1999; Sena et al., 2009; Ubeda-Tomas et al., 2008). As the
meristem size of bri1 is smaller than that of wild type (Fig. 2A,B,
left-most panels), we normalized the number of CycB1;1-GFPexpressing cells to the number of meristematic cells. This
normalization represents the proportion of meristematic cells that
are present at the G2-M phase of the cell cycle (Table 1) (Dello
Ioio et al., 2007). In bri1 lines, or in wild-type lines treated with
BRZ, the fraction of cells at the G2-M phase was reduced by ~40%
(Fig. 3A,B,C; Table 1). In addition, we performed quantitative
reverse-transcriptase (qRT-PCR) expression analysis of CYCB1;1
and the cytokinesis marker KNOLLE (Lauber et al., 1997). In this
case, in order to overcome the difference in root length between
samples, we normalized their expression level to RCH1 transcript
(Casamitjana-Martinez et al., 2003) (Fig. 3E). RCH1 served us as
reference for meristem size as it is specifically expressed in
Col-0
bri1
mock
BRZ
Cyclin-expressing cells (x)
Meristem cell number* (y)
Proportion of meristematic cells
present at the G2-M phase† (x/y)
12.6±0.78
5.1±0.67‡
19.2±1.26
10.7±0.78‡
32.1±0.62
23.5±1.97‡
27.8±0.82
25±0.48‡
0.39±0.02
0.22±0.03‡
0.69±0.04
0.43±0.03‡
*Root-meristem cell number is expressed as the number of cortical cells in the meristem.
†
The proportion of meristematic cells present at the G2-M phase is calculated by dividing the number of cells expressing CycB1;1-GFP with the root-meristem cell number
(Dello Ioio et al., 2007).
Results are presented as mean±s.e.m.
‡
P≤0.05: Col-0 compared with bri1; mock treatment compared with BRZ treatment.
DEVELOPMENT
Table 1. Cell-division marker in bri1 lines and after BRZ treatment
842
RESEARCH ARTICLE
Development 138 (5)
Table 2. Summary of the genotypes harboring BRI1-GFP
Background
Promoter
pBRI1
pGL2
pSCR
pSHR
pRCH1
Col-0
bri1
pBRI1-BRI1-GFPox
bri1;pGL2-BRI1-GFP
bri1;pSCR-BRI1-GFP
bri1;pSHR-BRI1-GFP
bri1;pRCH1-BRI1-GFP
Fig. 3. BRs are required for normal cell cycle activity.
(A,B)Representative confocal laser scanning microscope image showing
the expression of CycB1;1-GFP, a G2/M phase marker. The images are
false-colored to indicate GFP (green) and PI (red). (A)bri1;CycB1;1-GFP
compared with CycB1;1-GFP in Col-0 background. (B)Seven-day-old
CycB1;1-GFP seedlings grown in the absence (left) or presence (right) of
3M BRZ. Broken lines mark borders between apical and basal meristem
(lower line), and between the basal meristem and the EDZ (upper line). In
wild type, the basal meristem exceeds the captured image field. Scale
bar: 50m. (C)The number of meristem cells expressing CycB1;1-GFP
divided by the number of meristematic cortical cells. There is a drop in the
proportion of meristematic cells expressing CycB1;1-GFP in bri1 when
compared with Col-0 (Table 1). Results are presented as mean±s.e.m.
(D)Confocal laser scanning microscope images of roots subjected to
immunofluorescence analysis with anti-KNOLLE. Scale bar: 50 m.
(E) qRT-PCR analysis, demonstrating the expression level of CYCB1;1 and
KNOLLE relative to RCH1which represents ‘total meristematic cells’.
CYCB1;1 and KNOLLE transcripts are reduced in the absence BR
signaling. (F-H)qRT-PCR analysis on Col-0 plants treated with BL or BRZ,
demonstrates that impaired cell progression in bri1 is not associated with
changes in the expression level of PIN1, PIN3 and PIN7. Data are
mean±s.e.m. (I,J)Confocal laser scanning microscope images of pPIN1PIN1-GFP roots (I) and roots subjected to immunofluorescence analysis
with anti-PIN7 (J). Scale bars: 50m.
meristematic cells and is not modulated by BRs (see Fig. S2 in the
supplementary material). The results show that in agreement with
the reduced cell cycle activity, the expression of CYCB1;1 and
KNOLLE was reduced in bri1 by 50% when compared with wild
type (Fig. 3E). In addition, reduced immunofluorescence signal of
BRs are important to maintain gradual cell
progression in the meristem
We hypothesized that the reduced cell cycle activity in bri1 will be
associated with slow cell progression along the meristematic zones.
Therefore, we analyzed the effect of BRs in two consecutive zones
in the meristem, the apical and the basal meristem, using well
defined morphological criteria (Beemster et al., 2003; Ishikawa and
Evans, 1995; Verbelen et al., 2006; Zhang et al., 2010) (Fig. 1A).
Cells in the basal meristem are larger than cells in the apical
meristem (Fig. 2C); they cease dividing or divide at a very slow rate
and do not yet undergo fast elongation. Indeed, we did not observe
CycB1;1-GFP expression in the cortical cells of the basal meristem
(n74). These criteria were applied to both wild-type and bri1. bri1
had marginally fewer cells in the apical meristem and reduced
meristematic length compared with wild type, a difference that was
further accentuated in the basal meristem (Fig. 2A,B, middle and
right panels). Specifically, the length of the basal meristem of the
mutant was reduced by 60% and it had 40% fewer cells than wild
type. This is in contrast to a much smaller reduction in the apical
meristem size of the mutant and its corresponding cell number (34%
and 14%, respectively). Finally, we repeated our analysis in the
strong BR biosynthesis mutant cpd and observed a similar cellular
behavior; the cell number in the basal meristem was more affected
than in wild type of its own mutant background, whereas no
significant drop in cell number was observed in the apical meristem
(see Fig. S1B in the supplementary material). The dramatic drop in
cell number in the basal meristem relative to the apical meristem
suggests that bri1 cells fail to progress normally to the basal
meristem.
Failure of cells to progress along the meristem could be also
explained by slow or impaired cell expansion. We therefore
calculated the average cell size in the apical and basal meristem of
bri1. As shown in Fig. 2C, BRs affected cell expansion in the
DEVELOPMENT
KNOLLE in response to BRZ treatment is consistent with a
decrease in transcript levels (Fig. 3D). We therefore conclude that
BRs are required for normal cell cycle activity.
In addition to impaired cell cycle activity, stem cell niche
malfunction could also affect cell number in the root meristem
(Dello Ioio et al., 2007; Scheres, 2007). We therefore grew lines of
Arabidopsis with stem cell niche patterning markers in the presence
and absence of BRZ (see Fig. S3 in the supplementary material).
Among six markers tested, the fluorescence signal corresponding
to pPLT1-erCFP and pWOX5-GFP was slightly reduced by BRZ
treatment, and pAGL42-GFP signal showed a dramatic reduction
(see Fig. S3A,B,D in the supplementary material; Fig. 6A; see Fig.
S9A in the supplementary material). WOX5 promotes columella
stem cell (CSC) fate in the distal meristem (Sarkar et al., 2007).
Indeed, its moderate modification by low BR level is in agreement
with the reduction in the CSC frequency observed in bri1, as
reported and discussed in the accompanying paper by GonzálezGarcía et al. (González-García et al., 2011).
Epidermal BRI1 controls root growth
RESEARCH ARTICLE
843
meristem, and the difference in cell size between wild-type and
bri1 cells was more severe as cells progressed along the distinct
zones. These results indicate that cells fail to expand normally in
the meristem. Taken together, BRs promote mitotic cell cycle
activity and cell expansion, thus ensuring the normal cell
progression along the meristematic zones.
BR-mediated control of meristem size does not
involve changes in the expression level of the
stele-localized PIN1, PIN3 and PIN7
We next asked whether BR activity in the root meristem is
associated with changes in the expression level of PIN1, PIN3 and
PIN7, which mediate the balance between cell proliferation and
differentiation (Dello Ioio et al., 2008). To achieve this, we
performed qRT-PCR expression analysis in BL and BRZ treated
roots (Fig. 3F-H). We detected no change in the transcript level of
these PINs in the absence of BR signal, similar to their unchanged
protein level (PIN1 and PIN7 is shown, Fig. 3I,J). In addition, short
and long BL treatment had no effect (Fig. 3F-H). Thus, BRmediated control of root meristem size occurs through a different
mechanism from that proposed by the auxin and cytokinin models
for promoting cell proliferation and differentiation, respectively.
Epidermal BRI1 activity promotes cell expansion
and cell proliferation
We have shown that BRs maintains normal cell progression in
the meristem by promoting cell expansion and proliferation (Figs
1-3). We therefore asked how these processes are affected by the
spatial activity of BRI1. BRI1 is expressed in all cell layers of
the root (see Fig. S4 in the supplementary material). To study the
spatiotemporal control of BR signaling in the root, we directed
the expression of BRI1 to specific cell files in bri1, using the
promoters pGL2, pSCR and pSHR (Helariutta et al., 2000; Lin
and Schiefelbein, 2001; Wysocka-Diller et al., 2000) (Table 2;
Fig. 1A; Fig. 4A). These promoters drive expression in the
epidermis, endodermis, and QC and stele cell files, respectively
(Fig. 4A). Expression driven by pGL2 in the epidermis is
confined to atrichoblast (epidermal non-hair-cell) and appears
also in cells belonging to the LRC (Fig. 4A; see Fig. S5A in the
supplementary material). To study the contribution of
meristematic BRI1 activity per se to root length, we also
expressed BRI1 under the RCH1 promoter. Because BRI1-GFP
expression in the transgenic lines exceeds endogenous BRI1
levels, we also used pBRI1-BRI1-GFPox (Table 2). This line is
known to cause ubiquitous BRI1 overexpression (Friedrichsen et
al., 2000).
We initially compared the rate of root growth and meristem size
of the different transgenic lines with wild type. The bri1;pRCH1BRI1-GFP line exhibited similar growth rate and slightly smaller
meristem when compared with wild type (Fig. 4C,D). Thus, BR
activity in the meristem determines meristem size.
The root length and root growth rate of bri1;pSHR-BRI1-GFP
and bri1;pSCR-BRI1-GFP lines was reduced compared with wild
type (Fig. 4B,C). In accordance, their meristem size was smaller
(Fig. 4D). By contrast, pBRI-BRI1-GFPox and bri1;pGL2-BRI1GFP had a similar growth rate to wild type (Fig. 4B,C). Strikingly,
bri1;pGL2-BRI1-GFP lines had bigger meristem than wild type
and pBRI-BRI1-GFPox (Fig. 4D). Thus, BR perception in the
epidermis is sufficient to control root length and meristem size.
This regulation does not appear to involve the activity of the BRI1
homologs BRL1 and BRL3, as the changes in their transcript levels
do not correlate with the observed phenotypes (see Fig. S6 in the
supplementary material) (Cano-Delgado et al., 2004; Zhou et al.,
2004).
It is intriguing that BRI1 overexpression in the outer cell file, as
in bri1;pGL2-BRI1-GFP, resulted in bigger meristem relative to
wild-type, whereas ubiquitous overexpression of BRI1, as in pBRIBRI1-GFPox, did not (Fig. 4D; Fig. 5). We reasoned that the
meristem phenotype in pBRI-BRI1-GFPox was a result of an
above-optimal BR response. To test our hypothesis, we performed
a sensitivity assay to BRZ (see Fig. S7 in the supplementary
material). As shown, pBRI-BRI1-GFPox line was more resistant to
the inhibitory effect of BRZ on root elongation when compared
with either wild-type or to bri1;pGL2-BRI1-GFP. We next asked
whether the short root of bri1;pSHR-BRI1-GFP and bri1;pSCRBRI1-GFP is a result of above-optimal BR activity in the inner cell
files that limits growth. To examine this possibility, we crossed
bri1;pGL2-BRI1-GFP with these lines. The resultant crosses
DEVELOPMENT
Fig. 4. Root growth in lines expressing
BRI1-GFP in specific cell files. (A)Confocal
laser scanning microscope images of Col-0,
bri1 and transgenic lines expressing BRI1-GFP
are shown. pBRI1-BRI1-GFPox is in Col-0
background. All other transgenic lines are in
bri1 background. BRI1-GFP is expressed under:
pRCH1 (meristem); pGL2 (LRC and
atrichoblasts); pSCR (endodermis and QC);
pSHR (stele). Scale bar: 50m. (B,C)Root
length in different days (B) and calculated
growth rate (C) of Col-0, bri1 and the
different transgenic lines. DAG, days after
germination. (D)Relative root meristem length
of Col-0, bri1 and the different transgenic
lines. (E)Mature cortical cell length. Sevenday-old seedlings were analyzed. Results are
presented as mean±s.e.m.
844
RESEARCH ARTICLE
Development 138 (5)
but they had similar cell size (Fig. 5C-F). These results suggest that
BRI1 activity in the epidermis is sufficient to promote cell
expansion and proliferation.
In agreement with the effect of the transgenic lines on cell
proliferation, the expression level of CYCB1;1 and KNOLLE
relative to RCH1 expression was lower when BRI1 was expressed
from the inner cell files, but similar to wild type when BRI1 was
expressed from the epidermis (Fig. 5G). This is in agreement with
CycB1;1-GFP expression in bri1;pGL2-BRI1-GFP and bri1;pSHRBRI1-GFP (Fig. 5H).
Taken together, the results shown in Figs 4 and 5 suggest that BR
signaling in the root epidermis is sufficient to control the gradual cell
progression in the meristem. In addition, the positive correlation
between cell size and cell proliferation in the different lines further
demonstrates that these processes are affected by BR activity.
exhibited long roots, similar to bri1;pGL2-BRI1-GFP (see Fig. S8
in the supplementary material). Thus, BRI1 activity in the inner cell
files does not restrict growth, and inhibition of root growth
probably depends on high BR activity in multiple cell files.
To examine how spatial BRI1 activity affects cell progression in
the meristem, we performed cellular analysis in the apical and basal
meristem (Fig. 5). bri1;pSHR-BRI1-GFP and bri1;pSCR-BRI1GFP seedlings had reduced size of both meristematic zones
compared with wild type (Fig. 5A,B). In addition, the number of
cells significantly dropped only in the basal meristem (Fig. 5C,D)
and cell expansion in the two zones was impaired (Fig. 5E,F). By
contrast, the size of the meristematic zones of bri1;pGL2-BRI1GFP lines and their corresponding cell number exceeded wild type,
The known BR signaling pathway, from BRI1 to
BES1 and BZR1, acts locally
BES1 and BZR1 are transcription factors known to operate
downstream in the BRI1 signaling pathway. To test whether the
signal from the epidermis to the inner-cell files requires their
activity, we examined the BRRE element-containing BRox2
(CYP85A2) gene. BRox2 is a BR-biosynthesis gene whose
expression level is rapidly negatively modulated by BR treatment
and whose basal expression level is higher in the stele when
compared with the epidermis and LRC (Fig. 6D) (Birnbaum et al.,
2003; He et al., 2005). In bri1;pSHR-BRI1-GFP roots, BRox2 basal
expression level (mock) was lower than wild type.
Hence, the BR pathway is highly active in the stele of
bri1;pSHR-BRI1-GFP lines (Fig. 6D, right panel). In agreement,
BRox2 expression level decreased in response BL treatment,
ultimately reaching lower transcript level than BL-treated wild
type. Low BR response resulted in high BRox2 expression levels
and bri1;pGL2-BRI1-GFP roots had high basal expression levels
of BRox2. Furthermore, bri1;pGL2-BRI1-GFP roots were less
sensitive to BL treatment when compared with wild type.
DEVELOPMENT
Fig. 5. Epidermal BRI1 activity promotes cell proliferation and cell
expansion in the root meristem. (A,B)Relative length of
meristematic zones. (C,D)Relative number of cortical cells. (E,F)Relative
size of cortical cells (calculated as meristem length divided by the
cortical-cell number). (A,C,E) Quantification of apical meristem. (B,D,F)
Quantification of basal meristem. The relative measurements are given
as the percentage of Col-0 plants. Seven-day-old seedlings were
analyzed. Results are presented as mean±s.e.m. (G)qRT-PCR analysis
demonstrating the expression level of CYCB1;1 and KNOLLE relative to
RCH1, which represents ‘total meristematic cells’. All genes were first
normalized to the endogenous control. (H)Representative confocal
laser scanning microscope image showing the expression of CycB1;1GFP.
The QC- and stele-enriched AGL42 is one target for
a BRI1-mediated signal that is initiated
exclusively in the epidermis
To provide molecular evidence for a signal from the epidermis to the
inner-cell files, we searched for genes that are specifically expressed
in the inner cells and are modulated by BR activity. One such
candidate was the QC marker AGL42 (Nawy et al., 2005) (Fig. 6A;
see Fig. S9A in the supplementary material). In agreement with the
pAGL42-GFP reporter line, AGL42 showed the expected tissuespecific expression pattern [QC (which is included in the endodermis
data set)] and stele (Fig. 6A,B; see Fig. S9A in the supplementary
material) (Birnbaum et al., 2003). AGL42 transcript is dramatically
reduced in BRZ-treated seedlings and in bri1, but we detected no
change in its expression level in response to short BL treatment and
a moderate increase after longer BL treatment (Fig. 6C). Therefore,
we examined its expression in the different lines. Strikingly, AGL42
had lower expression level in bri1;pSCR-BRI1-GFP and bri1;pSHRBRI1-GFP, similar to bri1. By contrast, bri1;pGL2-BRI1-GFP lines
exhibited high AGL42 expression compared with wild type. Hence,
direct BRI1 activity in the QC and stele, where AGL42 transcript is
normally enriched, did not modulate its expression. Instead, the
expression of AGL42 was restored by BRI1-mediated signal that
originates in the epidermis. Thus, we provide molecular evidence
that BRI1, specifically expressed in the epidermis, controls gene
expression in the inner cell files.
Epidermal BRI1 controls root growth
RESEARCH ARTICLE
845
Thus, as opposed to AGL42, BRI1 expression in the epidermis
does not appear to affect early BR target in the stele (Fig. 6D). As
a control, BES1 is not seen to move from the epidermis to the inner
cells in roots (see Fig. S9B in the supplementary material).
Next, we tested whether BES1 and BZR1 activity in the stele
could regulate genes in the epidermis. WAG2 is a BRRE elementcontaining gene expressed in the epidermis (Fig. 6E, left panel).
WAG2 is positively regulated by BRs. Indeed, the basal level of
WAG2 was higher in bri1;pGL2-BRI1-GFP and lower in
bri1;pSHR-BRI1-GFP roots when compared with wild type. After
BL treatment, WAG2 expression in bri1;pGL2-BRI1-GFP and
bri1;pSHR-BRI1-GFP reached higher and lower levels,
respectively, when compared with wild type (Fig. 6E, right panel).
Thus, BES1/BZR1 activity in the stele does not appear to regulate
their early target in the epidermis.
To examine the expression of early BR-response genes further, we
established a pDWF4-GUS reporter line. The DWF4 promoter
contains the BRRE element and is a direct target of BES1/BZR1 (He
et al., 2005). In wild type, the DWF4 promoter fragment drives low
GUS expression in the epidermis and LRC (Fig. 6F). We therefore
crossed the pDWF4-GUS reporter line to bri1 and the transgenic
lines. As expected, the GUS signal was remarkably increased in bri1
when compared with wild type, but remained confined to the
epidermis (Fig. 6G; see Fig. S9C in the supplementary material).
High level of GUS expression was also detected in bri1;pSCR-BRI1GFP and bri1;pSHR-BRI1-GFP (Fig. 6I,J). As shown in Fig. 6K, the
GUS signal was observed only in the epidermis and LRC, similar to
its pattern in the bri1 background (see Fig. S9C in the supplementary
material). Hence, BRI1 activity in the inner cell files does not
regulate BES1/BZR1 targets in the epidermis, in agreement with the
qRT-PCR data (Fig. 6E). In bri1;pGL2-BRI1-GFP, no GUS signal
was detected in the epidermis, except for relative high levels in some
columella cells when compared with wild type (Fig. 6H).
Taken together, we conclude that BES1and BZR1 act locally,
suggesting that the signal from the epidermis to the inner cells is
transmitted by a different component.
DISCUSSION
This study provides an explanation for the short-root phenotype of
bri1 and uncovers a distinct mode of meristem-size control when
compared with other hormones. Specifically, our work
demonstrates that BRs are necessary to maintain a coherent
DEVELOPMENT
Fig. 6. BRI1-mediated signal, from the epidermis
to the inner cells, as opposed to local BR
response. (A)Confocal laser scanning microscope
image showing pAGL42-GFP in 7-day-old Col-0
seedlings mock treated (left) or treated with 3M
BRZ (right). Cell borders are marked by PI (red). Scale
bar: 20m. (B)Cell type-specific expression of the
MADS-BOX gene AGL42 (At5g62165). (C)qRT-PCR
expression analysis showing the relative expression
level of AGL42 in the different transgenic lines.
(D,E)qRT-PCR expression analysis. Cell type-specific
expression of BROX2 (At3g30180) (D, left panel)
and WAG2 (At3g14370) (E, left panel) correlates
with their relative expression level in the different
lines (D,E, right panel). Data for the cell typespecific expression are derived from Birnbaum
et al. (Birnbaum et al., 2003) using
https://www.genevestigator.com/gv/index.jsp. qRTPCR analysis was performed on 6-day-old root tips
that were mock treated, treated with 100 nM BL for
3 hours or grown in the presence of 2M BRZ.
Expression level is normalized to mock-treated Col-0
control. Data are mean±s.e.m. of three independent
experiments. (F-K)Epidermal expression of pDWF4GUS is regulated locally. GUS signal is low in the
epidermis of wild type (F) when compared with the
high signal in the epidermis of bri1 (G, see also Fig.
S9 in the supplementary material) or when BRI1 is
expressed in the inner cell-layers (I-K). Root cell files
are marked as in Fig. 1A. Scale bars: 50 m.
846
RESEARCH ARTICLE
Development 138 (5)
Fig. 7. Model for BR-mediated control of root meristem size. (A)Reaching a critical cell size is an essential requirement for cell cycle
progression, and BRs positively affect both processes. In bri1, cells fail to expand normally and have impaired cell cycle activity. These defects
probably impinge on their ability to enter the mitotic phase and delay their progression from the apical to the basal meristem. This is further
reflected in the relative number of cells in the two zones (represented by the colored columns). Cells are represented by gray boxes and cytokinesis
by dashed lines. (B)BR signaling in the epidermis is sufficient to control root meristem size, whereas BR signaling in the inner cell files (i.e. stele) is
not. This indicates that the inner cells must receive instructive signal(s) initiated by BRI1 exclusively in the epidermis. The MADS-BOX gene AGL42 is
a target of such a signal. The signal from the epidermis to the inner cells is transmitted by a different component from BES1 and BZR1, which
regulate their direct target genes (i.e. BR-biosynthesis genes) locally in cells that express BRI1.
BRs promote cell expansion rate and mitotic cycle
to maintain gradual cell progression in the
meristem
We propose that BRs are required to drive specific stages of the cell
cycle, probably before cell entry into the mitosis. Our assumption
is based on the observation that the proportion of cells present at
the G2-M phase and cytokinesis is lower in bri1 when compared
with wild type (Fig. 3A-D). In addition, the number of cells in the
apical meristem is less affected when compared with the severe
reduction in cell number in the basal meristem. Hence, cell cycle
progression is retained. Our assumption is also consistent with the
ability of high CycD3;1 level to suppress the reduced number of
cells in bri1, as reported in the accompanying paper by GonzálezGarcía et al. (González-García et al., 2011).
Does BR regulate the cell cycle machinery per se? Thus far, BRs
have been reported to induce the expression level of two core cell
cycle genes: CycD3;1 in suspension cells and CDKB1;1 in darkgrown seedlings (Hu et al., 2000; Yoshizumi et al., 1999).
However, these genes have not been shown to be early targets of
BRs. Moreover, in root meristematic cells, the transcript level of
CycD3;1 is not affected by short and long BL treatment and by
BRZ treatment (Y.H., unpublished). Likewise, CYCB1;1 and
KNOLLE expression level is not responding to short BL application
(Fig. 3E). Thus, whether BRs directly affect core cell cycle genes
at the transcriptional or post-transcriptional level is currently an
unanswered question.
It is well established that cell division and cell size are
coordinated, and cell growth is an essential requirement for cell
cycle progression (Jorgensen and Tyers, 2004; Martin and
Berthelot-Grosjean, 2009; Moseley et al., 2009). It is plausible that
defects which determine how fast cells will double their size before
division restrain cell cycle progression in bri1. Our data show that
BRs are necessary to promote cell expansion rate and maintain
normal cell size along the distinct root zonation (Fig. 1E; Fig. 2C).
The size of cells in the meristem is a result of increasing
cytoplasmic volume (Beemster et al., 2003). Although we do not
exclude the involvement of BRs in the increase of cell-biomass, the
role of BRs in regulating expansion processes through cell-wall
modification is evident (Zurek et al., 1994). In addition, short BR
treatment is known to positively regulate the expression of cell-wall
organization enzymes required for cell expansion and division
(Nemhauser et al., 2004).
BR activity represents an additional pathway for
hormonal control of root meristem-size
Depending on their relative level, cytokinin and auxin promote
cell differentiation and proliferation, respectively (Dello Ioio et al.,
2008). Auxin gradients can prolong or shorten the distinct phases
of proliferation and differentiation, and PIN proteins are essential
for these processes (Blilou et al., 2005; Grieneisen et al., 2007).
Cytokinin activity ultimately reduces the expression of PIN1,
PIN3 and PIN7 in the stele to counteract auxin-positive regulation
on cell division and initiate differentiation (Dello Ioio et al., 2008).
Our data shows that BR activity is not associated with changes in
the expression level of these stele-localized PINs (Fig. 3).
Furthermore, cellular analysis indicates that in the presence of
cytokinin, the root meristem has reduced cell number although the
relative proportion of cells at the G2-M phase is not affected
(Dello Ioio et al., 2007). The relative low proportion of cells at the
G2-M phase in bri1 further argues against BR role in
cytokinin/auxin interplay for determination of cell differentiation
(Fig. 3). Positive interaction between BRs and auxin in promoting
DEVELOPMENT
developmental gradient of cells in the meristem. This dynamics
depends on BR-positive effect on cell cycle activity and cellexpansion rate. Remarkably, root meristem-size is controlled by
BRI1 activation in the epidermis, suggesting that the spatial
regulation of both above- and below-ground organs may share
common mechanistic principles. In the epidermis, BRI1 activity
induces signal(s) to communicate with the inner cells. This signal
is transmitted by a different component than BES1 and BZR1,
which act locally (Fig. 7).
root growth is evident through the action of BRX, which
maintains optimal BR levels for auxin responses (Mouchel et al.,
2006).
Remarkably, hormonal control of root meristem size can occur
from a subset of cells. Hence, as opposed to cytokinin and GAs,
which act in the transition zone of the stele and endodermis,
respectively, we demonstrate that BRs are required in the epidermis
(Dello Ioio et al., 2007; Ubeda-Tomas et al., 2009; Ubeda-Tomas
et al., 2008).
The role of epidermal BRI1 activity in controlling
meristem size
Our work clearly demonstrates that BRI1 activity in the epidermis
promotes root meristem size. By contrast, BR-signal from the inner
cell files (endodermis/QC and stele) had a lesser effect. Thus, BRs
exert similar spatial regulation in both root and shoot (SavaldiGoldstein and Chory, 2008; Savaldi-Goldstein et al., 2007). The
simple organization of the root meristem combined with available
cell type-specific gene expression data and reporter genes, allowed
us to characterize the mode of BR activity and whether the inner
cells in the meristem receive a signal from the epidermis.
Here, we provide evidence that such a signal is present. Hence,
the expression of the MADS-box gene AGL42 depends on BRmediated signal from the epidermis and not from the QC,
endodermis and the stele (Fig. 6). The requirement of MADS-BOX
genes for normal root development has been recently established.
However, the exact developmental role of AGL42 is currently
unknown (Moreno-Risueno et al., 2010; Nawy et al., 2005; TapiaLopez et al., 2008). Taken together, it is apparent that the activity
of hormones in selected cells of the plant body can regulate the
growth of the whole organ. Our work further supports the
importance of cell-cell communication as a mechanism for
controlling meristem size.
Acknowledgements
We appreciate the technical assistance of M. Rosenberg, S. Nissani and M.
Duvshani-Eshet at the LSE infrastructure unit. We thank G. Beemster for
fruitful discussions, and N. Geldner, J. Long, R. Fluhr and B. Podbilewicz for
comments on the manuscript. We also thank S. Sabatini, B. Scheres, P. Benfey
and J. Long for providing seeds and promoter fragments. We are grateful to K.
Palme for the gift of PIN7 antibodies. Y.H. is supported by a Fine Fellowship.
This work was supported by a grant from the NSF to J.C. (IOS-06-49389), by
the Howard Hughes Medical Institute, and by research grants to S.S.-G. from
FP7-PEOPLE-IRG-2008 and the Israel Science Foundation (Research Grant
Awards No. 1498/09 and 1603/09). Deposited in PMC for release after 6
months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.061804/-/DC1
References
Achard, P., Gusti, A., Cheminant, S., Alioua, M., Dhondt, S., Coppens, F.,
Beemster, G. T. and Genschik, P. (2009). Gibberellin signaling controls cell
proliferation rate in Arabidopsis. Curr. Biol. 19, 1188-1193.
Beemster, G. T. and Baskin, T. I. (1998). Analysis of cell division and elongation
underlying the developmental acceleration of root growth in Arabidopsis
thaliana. Plant Physiol. 116, 1515-1526.
Beemster, G. T. and Baskin, T. I. (2000). Stunted plant 1 mediates effects of
cytokinin, but not of auxin, on cell division and expansion in the root of
Arabidopsis. Plant Physiol. 124, 1718-1727.
Beemster, G. T., Fiorani, F. and Inze, D. (2003). Cell cycle: the key to plant
growth control? Trends Plant Sci. 8, 154-158.
Benkova, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertova, D.,
Jurgens, G. and Friml, J. (2003). Local, efflux-dependent auxin gradients as a
common module for plant organ formation. Cell 115, 591-602.
RESEARCH ARTICLE
847
Birnbaum, K., Shasha, D. E., Wang, J. Y., Jung, J. W., Lambert, G. M.,
Galbraith, D. W. and Benfey, P. N. (2003). A gene expression map of the
Arabidopsis root. Science 302, 1956-1960.
Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., Heidstra,
R., Aida, M., Palme, K. and Scheres, B. (2005). The PIN auxin efflux facilitator
network controls growth and patterning in Arabidopsis roots. Nature 433, 3944.
Cano-Delgado, A., Yin, Y., Yu, C., Vafeados, D., Mora-Garcia, S., Cheng, J. C.,
Nam, K. H., Li, J. and Chory, J. (2004). BRL1 and BRL3 are novel
brassinosteroid receptors that function in vascular differentiation in Arabidopsis.
Development 131, 5341-5351.
Casamitjana-Martinez, E., Hofhuis, H. F., Xu, J., Liu, C. M., Heidstra, R. and
Scheres, B. (2003). Root-specific CLE19 overexpression and the sol1/2
suppressors implicate a CLV-like pathway in the control of Arabidopsis root
meristem maintenance. Curr. Biol. 13, 1435-1441.
Clouse, S. D. and Sasse, J. M. (1998). Brassinosteroids: Essential regulators of
plant growth and development. Ann. Rev. Plant Physiol. Plant Mol. Biol. 49, 427451.
Colon-Carmona, A., You, R., Haimovitch-Gal, T. and Doerner, P. (1999).
Technical advance: spatio-temporal analysis of mitotic activity with a labile cyclinGUS fusion protein. Plant J. 20, 503-508.
Dello Ioio, R., Linhares, F. S., Scacchi, E., Casamitjana-Martinez, E., Heidstra,
R., Costantino, P. and Sabatini, S. (2007). Cytokinins determine Arabidopsis
root-meristem size by controlling cell differentiation. Curr. Biol. 17, 678-682.
Dello Ioio, R., Nakamura, K., Moubayidin, L., Perilli, S., Taniguchi, M.,
Morita, M. T., Aoyama, T., Costantino, P. and Sabatini, S. (2008). A genetic
framework for the control of cell division and differentiation in the root
meristem. Science 322, 1380-1384.
Friedrichsen, D. M., Joazeiro, C. A., Li, J., Hunter, T. and Chory, J. (2000).
Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat
receptor serine/threonine kinase. Plant Physiol. 123, 1247-1256.
Galinha, C., Hofhuis, H., Luijten, M., Willemsen, V., Blilou, I., Heidstra, R.
and Scheres, B. (2007). PLETHORA proteins as dose-dependent master
regulators of Arabidopsis root development. Nature 449, 1053-1057.
Gleave, A. P. (1992). A versatile binary vector system with a T-DNA organisational
structure conducive to efficient integration of cloned DNA into the plant
genome. Plant Mol. Biol. 20, 1203-1207.
González-García, M.-P., Vilarrasa-Blasi, J., Zhiponova, M., Divol, F., MoraGarcía, S., Russinova, E. and Caño-Delgado, A. I. (2011). Brassinosteroids
control meristem size by promoting cell cycle progression in Arabidopsis roots.
Development 138, 849-859.
Grieneisen, V. A., Xu, J., Maree, A. F., Hogeweg, P. and Scheres, B. (2007).
Auxin transport is sufficient to generate a maximum and gradient guiding root
growth. Nature 449, 1008-1013.
He, J. X., Gendron, J. M., Sun, Y., Gampala, S. S., Gendron, N., Sun, C. Q. and
Wang, Z. Y. (2005). BZR1 is a transcriptional repressor with dual roles in
brassinosteroid homeostasis and growth responses. Science 307, 1634-1638.
He, Z., Wang, Z. Y., Li, J., Zhu, Q., Lamb, C., Ronald, P. and Chory, J. (2000).
Perception of brassinosteroids by the extracellular domain of the receptor kinase
BRI1. Science 288, 2360-2363.
Helariutta, Y., Fukaki, H., Wysocka-Diller, J., Nakajima, K., Jung, J., Sena, G.,
Hauser, M. T. and Benfey, P. N. (2000). The SHORT-ROOT gene controls radial
patterning of the Arabidopsis root through radial signaling. Cell 101, 555-567.
Hu, Y., Bao, F. and Li, J. (2000). Promotive effect of brassinosteroids on cell
division involves a distinct CycD3-induction pathway in Arabidopsis. Plant J. 24,
693-701.
Ishikawa, H. and Evans, M. L. (1995). Specialized zones of development in roots.
Plant Physiol. 109, 725-727.
Jorgensen, P. and Tyers, M. (2004). How cells coordinate growth and division.
Curr. Biol. 14, R1014-R1027.
Kim, T. W., Guan, S., Sun, Y., Deng, Z., Tang, W., Shang, J. X., Sun, Y.,
Burlingame, A. L. and Wang, Z. Y. (2009). Brassinosteroid signal transduction
from cell-surface receptor kinases to nuclear transcription factors. Nat. Cell Biol.
11, 1254-1262.
Lauber, M. H., Waizenegger, I., Steinmann, T., Schwarz, H., Mayer, U.,
Hwang, I., Lukowitz, W. and Jurgens, G. (1997). The Arabidopsis KNOLLE
protein is a cytokinesis-specific syntaxin. J. Cell Biol. 139, 1485-1493.
Li, J. and Chory, J. (1997). A putative leucine-rich repeat receptor kinase involved
in brassinosteroid signal transduction. Cell 90, 929-938.
Lin, Y. and Schiefelbein, J. (2001). Embryonic control of epidermal cell patterning
in the root and hypocotyl of Arabidopsis. Development 128, 3697-3705.
Malamy, J. E. and Benfey, P. N. (1997). Organization and cell differentiation in
lateral roots of Arabidopsis thaliana. Development 124, 33-44.
Martin, S. G. and Berthelot-Grosjean, M. (2009). Polar gradients of the DYRKfamily kinase Pom1 couple cell length with the cell cycle. Nature 459, 852-856.
Moreno-Risueno, M. A., Van Norman, J. M., Moreno, A., Zhang, J., Ahnert,
S. E. and Benfey, P. N. (2010). Oscillating gene expression determines
competence for periodic Arabidopsis root branching. Science 329, 1306-1311.
Moseley, J. B., Mayeux, A., Paoletti, A. and Nurse, P. (2009). A spatial gradient
coordinates cell size and mitotic entry in fission yeast. Nature 459, 857-860.
DEVELOPMENT
Epidermal BRI1 controls root growth
RESEARCH ARTICLE
Mouchel, C. F., Briggs, G. C. and Hardtke, C. S. (2004). Natural genetic variation
in Arabidopsis identifies BREVIS RADIX, a novel regulator of cell proliferation and
elongation in the root. Genes Dev. 18, 700-714.
Mouchel, C. F., Osmont, K. S. and Hardtke, C. S. (2006). BRX mediates
feedback between brassinosteroid levels and auxin signalling in root growth.
Nature 443, 458-461.
Muller, A., Guan, C., Galweiler, L., Tanzler, P., Huijser, P., Marchant, A., Parry,
G., Bennett, M., Wisman, E. and Palme, K. (1998). AtPIN2 defines a locus of
Arabidopsis for root gravitropism control. EMBO J. 17, 6903-6911.
Nakamura, A., Fujioka, S., Sunohara, H., Kamiya, N., Hong, Z., Inukai, Y.,
Miura, K., Takatsuto, S., Yoshida, S., Ueguchi-Tanaka, M. et al. (2006). The
role of OsBRI1 and its homologous genes, OsBRL1 and OsBRL3, in rice. Plant
Physiol. 140, 580-590.
Nakaya, M., Tsukaya, H., Murakami, N. and Kato, M. (2002). Brassinosteroids
control the proliferation of leaf cells of Arabidopsis thaliana. Plant Cell Physiol.
43, 239-244.
Nawy, T., Lee, J. Y., Colinas, J., Wang, J. Y., Thongrod, S. C., Malamy, J. E.,
Birnbaum, K. and Benfey, P. N. (2005). Transcriptional profile of the
Arabidopsis root quiescent center. Plant Cell 17, 1908-1925.
Nemhauser, J. L., Mockler, T. C. and Chory, J. (2004). Interdependency of
brassinosteroid and auxin signaling in Arabidopsis. PLoS Biol. 2, E258.
Perez-Perez, J. M., Ponce, M. R. and Micol, J. L. (2002). The UCU1 Arabidopsis
gene encodes a SHAGGY/GSK3-like kinase required for cell expansion along the
proximodistal axis. Dev. Biol. 242, 161-173.
Petricka, J. J. and Benfey, P. N. (2008). Root layers: complex regulation of
developmental patterning. Curr. Opin. Genet. Dev. 18, 354-361.
Reinhardt, B., Hanggi, E., Muller, S., Bauch, M., Wyrzykowska, J., Kerstetter,
R., Poethig, S. and Fleming, A. J. (2007). Restoration of DWF4 expression to
the leaf margin of a dwf4 mutant is sufficient to restore leaf shape but not size:
the role of the margin in leaf development. Plant J. 52, 1094-1104.
Ruzicka, K., Simaskova, M., Duclercq, J., Petrasek, J., Zazimalova, E., Simon,
S., Friml, J., Van Montagu, M. C. and Benkova, E. (2009). Cytokinin
regulates root meristem activity via modulation of the polar auxin transport.
Proc. Natl. Acad. Sci. USA 106, 4284-4289.
Sarkar, A. K., Luijten, M., Miyashima, S., Lenhard, M., Hashimoto, T.,
Nakajima, K., Scheres, B., Heidstra, R. and Laux, T. (2007). Conserved
factors regulate signalling in Arabidopsis thaliana shoot and root stem cell
organizers. Nature 446, 811-814.
Savaldi-Goldstein, S. and Chory, J. (2008). Growth coordination and the shoot
epidermis. Curr. Opin. Plant Biol. 11, 42-48.
Savaldi-Goldstein, S., Peto, C. and Chory, J. (2007). The epidermis both drives
and restricts plant shoot growth. Nature 446, 199-202.
Savaldi-Goldstein, S., Baiga, T. J., Pojer, F., Dabi, T., Butterfield, C., Parry, G.,
Santner, A., Dharmasiri, N., Tao, Y., Estelle, M. et al. (2008). New auxin
analogs with growth-promoting effects in intact plants reveal a chemical strategy
to improve hormone delivery. Proc. Natl. Acad. Sci. USA 105, 15190-15195.
Scheres, B. (2007). Stem-cell niches: nursery rhymes across kingdoms. Nat. Rev.
Mol. Cell Biol. 8, 345-354.
Sena, G., Wang, X., Liu, H. Y., Hofhuis, H. and Birnbaum, K. D. (2009). Organ
regeneration does not require a functional stem cell niche in plants. Nature 457,
1150-1153.
Stepanova, A. N., Hoyt, J. M., Hamilton, A. A. and Alonso, J. M. (2005). A
Link between ethylene and auxin uncovered by the characterization of two rootspecific ethylene-insensitive mutants in Arabidopsis. Plant Cell 17, 2230-2242.
Development 138 (5)
Swarup, R., Friml, J., Marchant, A., Ljung, K., Sandberg, G., Palme, K. and
Bennett, M. (2001). Localization of the auxin permease AUX1 suggests two
functionally distinct hormone transport pathways operate in the Arabidopsis
root apex. Genes Dev. 15, 2648-2653.
Swarup, R., Kramer, E. M., Perry, P., Knox, K., Leyser, H. M., Haseloff, J.,
Beemster, G. T., Bhalerao, R. and Bennett, M. J. (2005). Root gravitropism
requires lateral root cap and epidermal cells for transport and response to a
mobile auxin signal. Nat. Cell Biol. 7, 1057-1065.
Swarup, R., Perry, P., Hagenbeek, D., Van Der Straeten, D., Beemster, G. T.,
Sandberg, G., Bhalerao, R., Ljung, K. and Bennett, M. J. (2007). Ethylene
upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of
root cell elongation. Plant Cell 19, 2186-2196.
Szekeres, M., Nemeth, K., Koncz-Kalman, Z., Mathur, J., Kauschmann, A.,
Altmann, T., Redei, G. P., Nagy, F., Schell, J. and Koncz, C. (1996).
Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450,
controlling cell elongation and de-etiolation in Arabidopsis. Cell 85, 171182.
Tapia-Lopez, R., Garcia-Ponce, B., Dubrovsky, J. G., Garay-Arroyo, A., PerezRuiz, R. V., Kim, S. H., Acevedo, F., Pelaz, S. and Alvarez-Buylla, E. R.
(2008). An AGAMOUS-related MADS-box gene, XAL1 (AGL12), regulates root
meristem cell proliferation and flowering transition in Arabidopsis. Plant Physiol.
146, 1182-1192.
Ubeda-Tomas, S., Swarup, R., Coates, J., Swarup, K., Laplaze, L., Beemster,
G. T., Hedden, P., Bhalerao, R. and Bennett, M. J. (2008). Root growth in
Arabidopsis requires gibberellin/DELLA signalling in the endodermis. Nat. Cell
Biol. 10, 625-628.
Ubeda-Tomas, S., Federici, F., Casimiro, I., Beemster, G. T., Bhalerao, R.,
Swarup, R., Doerner, P., Haseloff, J. and Bennett, M. J. (2009). Gibberellin
signaling in the endodermis controls Arabidopsis root meristem size. Curr. Biol.
19, 1194-1199.
Verbelen, J. P., De Cnodder, T., Le J., Vissenberg, K. and Baluska, F. (2006).
The root apex of Arabidopsis thaliana consists of four distinct zones of growth
activities: meristematic zone, transition zone, fast elongation zone and growth
terminating zone. Plant Signal. Behav. 1, 296-304.
Wysocka-Diller, J. W., Helariutta, Y., Fukaki, H., Malamy, J. E. and Benfey, P.
N. (2000). Molecular analysis of SCARECROW function reveals a radial
patterning mechanism common to root and shoot. Development 127, 595603.
Yin, Y., Vafeados, D., Tao, Y., Yoshida, S., Asami, T. and Chory, J. (2005). A
new class of transcription factors mediates brassinosteroid-regulated gene
expression in Arabidopsis. Cell 120, 249-259.
Yoshizumi, T., Nagata, N., Shimada, H. and Matsui, M. (1999). An Arabidopsis
cell cycle -dependent kinase-related gene, CDC2b, plays a role in regulating
seedling growth in darkness. Plant Cell 11, 1883-1896.
Zhang, H., Han, W., De Smet, I., Talboys, P., Loya, R., Hassan, A., Rong, H.,
Jurgens, G., Paul Knox, J. and Wang, M. H. (2010). ABA promotes
quiescence of the quiescent centre and suppresses stem cell differentiation in
the Arabidopsis primary root meristem. Plant J. 64, 764-774.
Zhou, A., Wang, H., Walker, J. C. and Li, J. (2004). BRL1, a leucine-rich repeat
receptor-like protein kinase, is functionally redundant with BRI1 in regulating
Arabidopsis brassinosteroid signaling. Plant J. 40, 399-409.
Zurek, D. M., Rayle, D. L., McMorris, T. C. and Clouse, S. D. (1994).
Investigation of gene expression, growth kinetics, and wall extensibility during
brassinosteroid-regulated stem elongation. Plant Physiol. 104, 505-513.
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